Novel scaffold of adenylyl cyclase inhibitors for chronic pain and opioid dependence

ABSTRACT

The present invention relates to a method of treatment for chronic pain, opioid dependence, alcohol use disorder or autism using a class of pyrimidinone compounds, an adenylyl cyclase 1 (AC1) inhibitor. The invention described herein also pertains to pharmaceutical compositions and methods for treating diseases in mammals using those compounds disclosed herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This present patent application relates to and claims the prioritybenefit of U.S. Provisional Application Ser. No. 62/533,686, filed Jul.18 2017, the content of which is hereby incorporated herein by referencein its entirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under MH101673 awardedby the National Institute of Health. The government has certain rightsin the invention.

TECHNICAL FIELD

The present invention relates to a method of treatment for chronic pain,opioid dependence, alcohol use disorder or autism. Particularly, thepresent disclosure relates to a method of treatment for chronic pain,opioid dependence, alcohol use disorder or autism using a class ofpyrimidinone adenylyl cyclase inhibitors. The invention described hereinalso pertains to pharmaceutical compositions and methods for treatingdiseases in mammals using compounds disclosed herein.

BACKGROUND

This section introduces aspects that may help facilitate a betterunderstanding of the disclosure. Accordingly, these statements are to beread in this light and are not to be understood as admissions about whatis or is not prior art.

Chronic pain is a major health concern that costs the US more than $635billion per year (Gaskin and Richard, (2012) J. Pain 13:715-724). Inaddition to the financial impact, patients with chronic pain sufferextreme physical, emotional, and social burdens. For example,individuals often become socially isolated and confined to home as aresult of their chronic pain that is not well-controlled by today'savailable treatments. The drugs used for the management of chronic paininclude opioid analgesics, neuronal stabilizers such as anticonvulsants,and antidepressants. Opioids are the most widely used, and a recent NIHreport indicates that there are significant problems associated withlong-term opioid therapy for chronic pain (Volkow and McLellan (2016) NEngl J Med 374:1253-1263.). None of the agents provide sufficient reliefto allow patients to return to their normal activity level. Moreover,current pharmaceutical industry has retreated from studying novel paintherapeutics due to the enormous risk (Skolnick and Volkow (2016) Neuron92:294-297.). These observations indicate an essential need to identifynew agents acting on unique targets in the war on chronic pain.

Neurobiological, genetic, and preclinical studies have implicatedneuronal adenylyl cyclase type I (AC1) as a potential new drug target(Zhuo (2012) Drug Discov Today 17:573-582.). Adenylyl cyclases (AC) arean enzyme family that serve as effectors of numerous G protein coupledreceptors (e.g. opioid and dopamine receptors) and produce the secondmessenger cAMP from ATP (FIG. 1). Nine membrane-bound isoforms of ACshare a similar structure that includes an intracellular N-terminus,followed by two membrane-spanning domains alternating with twocytoplasmic (catalytic) domains that can be further divided into a and bregions (Sadana and Dessauer, (2009) Neurosignals 17:5-22.). The C1a andC2a domains make up the catalytic portion of the enzyme, and an X-raycrystal structure with the C1a domain from AC5 and the C2a domain fromAC2 was solved in 1997 (Tesmer et al., (1997) Science 278:1907-1916.).In contrast, no structural information exists regarding N-terminus, C1b,or C2b domains for any isoform. Each isoform is uniquely regulated by Gprotein α and βγ subunits, Ca²⁺, protein kinases, posttranslationalmodifications, and subcellular localization (Willoughby and Cooper,(2007) Physiol Rev 87:965-1010).

Group 1 ACs, represented by AC1, AC3, and AC8, are stimulated bycalmodulin in a Ca²⁺-dependent manner. Group 2 ACs are characterized bytheir conditional stimulation by Gβγ subunits and are represented byAC2, AC4, and AC7. AC2 and AC7 are also activated by protein kinase C.Group 3 ACs include AC5 and AC6, show robust negative regulation by Ga,subunits, and are also inhibited by submicromolar concentration of Ca²⁺as well as protein kinase A. Group 4 ACs contains only one member, AC9,which is unique among the ACs in being relatively insensitive toactivation by the small molecule diterpene, forskolin.

Membrane-bound ACs are highly expressed in the central nervous systemand generally have overlapping expression patterns (Sanabra and Mengod,(2011). J Chem Neuroanat 41:43-54.). Multiple AC isoforms are typicallyexpressed in individual cell types, making it difficult to elucidate thefunction(s) of individual isoforms in either native tissues or celllines. This problem has been addressed using a variety of recombinantapproaches, including overexpression, site-directed mutagenesis, and,most notably, global genetic deletions. These animals lacking one ormultiple AC isoforms have been essential tools to inform on thephysiological roles of AC signaling in the central nervous system(Sadana R et al., (2009). Neurosignals 17:5-22).

Physiological roles of AC1 and AC8: AC1 and AC8 are robustly activatedby Ca²⁺/calmodulin (Ca²⁺/CaM) and have overlapping expression patternsin neuronal tissues, including the hippocampus and several corticalregions (Defer N, et al., (2000). Am J Physiol Renal Physiol279:F400-F416). To explore their relative physiological roles, a numberof studies have been carried out with mice lacking either AC1(AC1^(−/−)), AC8 (AC8^(−/−)), or both isoforms (double knock out mice,DKO). Initial experiments with animals lacking Ca²⁺/CaM-stimulatedcyclases focused on long-term memory (LTM) and long-term potentiation(LTP) due to their high level of expression in the hippocampus. Theresults of these experiments implicated AC1 and AC8 in LTP and LTM(Ferguson and Storm, 2004). Importantly, it was found that long lastingLTP and memory deficits were marked in animals lacking both AC1 and AC8(DKO mice), but were mostly absent in animals deficient in only a singleAC isoform. However, a few studies found that AC1^(−/−) mice showedmodest deficits in other forms of LTP (Chen et al., (2014), Mol Pain10:65.), including a reduction in remote contextual fear memory that wasonly observed at a single time point. These observations clearlyimplicate Ca²⁺/CaM-stimulated cyclases in LTP and certain models ofmemory; however, selectively targeting a single AC isoform markedlyreduces the overall deficits. Furthermore, these findings also emphasizethe benefits of pharmacologically targeting overactive AC1 indose-dependent fashion versus complete inhibition or genetic deletion.

Additionally, AC1 knock out mice show less reward when given opioids andshow reduced symptoms of opioid dependence during withdrawal. Additionalreports suggest that AC linhibition may also provide a usefultherapeutic intervention for alcohol use disorder and autism (Bosse K Eet al., J. Pharmacol. Exp. Ther. 2017, 363 (2) 148-155; Sethna F., etal. Nat. Commun. 2017, 8, 14359).

Unfortunately, until now, the selective inhibition of ACs has not beenachieved, and simultaneous inhibition of multiple adenylyl cyclaseisoforms would likely result in significant adverse effects. There areunmet needs for better and safer medications targeting adenylyl cyclasesfor various therapeutic uses, including pain, opioid dependence, alcoholuse disorder and autism.

BRIEF SUMMARY OF INVENTION

In some embodiments, this invention relates to a method of treatingpain, opioid dependence, alcohol use disorder or autism comprising thestep of administering to a mammal in need of relief from said pain oropioid dependence thereof a therapeutically effective amount of one ormore compounds of a mammal in need thereof a therapeutically effectiveamount of one or more compounds of formula (I)

or a pharmaceutically acceptable salt thereof, and one or more carriers,diluents, or excipients, wherein

-   R₁ is hydrogen, halo, hydroxy, amino, nitro, cyano, thio, alkyl,    alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,    cycloalkyl, heterocyloalkyl, cycloalkenyl, heterocycloakenyl,    heterocyclyl, alkoxyl, alkylamino, alkylthio, haloalkyl, sulphonyl,    sulphonamide, alkylsulphonamide, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   R₂ is hydrogen, halo, hydroxy, amino, nitro, cyano, thio, alkyl,    alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,    cycloalkyl, heterocyloalkyl, cycloalkenyl, heterocycloakenyl,    heterocyclyl, alkoxyl, alkylamino, alkylthio, haloalkyl, sulphonyl,    sulphonamide, alkylsulphonamide, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   or R₁ and R₂ are taken together with the attached carbons to form an    optionally substituted cycle or heterocycle;-   R₃ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,    heteroalkynyl, cycloalkyl, heterocyloalkyl, cycloalkenyl,    heterocycloakenyl, heterocyclyl, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   R₄ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,    heteroalkynyl, cycloalkyl, heterocyloalkyl, cycloalkenyl,    heterocycloakenyl, heterocyclyl, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   or R₃ and R₄ are taken together with the attached carbons to form an    optionally substituted cycle or heterocycle;

and

-   R₅ is an alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,    heteroalkynyl, cycloalkyl, heterocyloalkyl, cycloalkenyl,    heterocycloakenyl, heterocyclyl, or an optionally substituted aryl,    arylalkyl, or arylalkenyl.

In some other embodiments, this invention relates to a method fortreating and management of a chronic pain.

In some other embodiments, this invention is related to a method fortreating and reducing pain and opioid dependence.

In some other embodiments, this invention is related to a method fortreating and reducing pain and opioid dependence further comprising thestep of administering a compound of formula I in combination with anopioid drug, wherein the compound of formula I enhances μ-opioidreceptor inhibition of adenylyl cyclase 1.

In some other embodiments, this invention is related to a method fortreating and reducing pain and opioid dependence, wherein the opioiddrug is selected from the group consisting of codeine, morphine,thebaine, oripavine, diacetylmorphine, nicomorphine,dipropanoylmorphine, diacetyldihydromorphine, acetylpropionylmorphine,desomorphine, methyldesorphine, dibenzoylmorphine, dihydrocodeine,ethylmorphine, heterocodeine, buprenorphine, etorphine, hydrocodone,hydromorphone, oxycodone, oxymorphone, fentanyl, alphamethylfentanyl,alfentanil, sufentanil, remifentanil, carfentanyl, ohmefentanyl,pethidine (meperidine), ketobemidone, desmethylprodine (MPPP),allylprodine, prodine, phenethylphenylacetoxypiperidine (PEPAP),promedol, propoxyphene, dextropropoxyphene, dextromoramide, bezitramide,piritramide, methadone, dipipanone, levomethadyl acetate (LAAM),difenoxin, diphenoxylate, loperamide, dezocine, pentazocine,phenazocine, buprenorphine, dihydroetorphine, etorphine, butorphanol,nalbuphine, levorphanol, levomethorphan, lefetamine, menthol,meptazinol, mitragynine, tilidine, tramadol, tapentadol, eluxadoline,nalmefene, naloxone, and naltrexone.

In some illustrative embodiments, the invention relates to a compound offormula (I)

or a pharmaceutically acceptable salt thereof, wherein

-   R₁ is hydrogen, halo, hydroxy, amino, nitro, cyano, thio, alkyl,    alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,    cycloalkyl, heterocyloalkyl, cycloalkenyl, heterocycloakenyl,    heterocyclyl, alkoxyl, alkylamino, alkylthio, haloalkyl, sulphonyl,    sulphonamide, alkylsulphonamide, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   R₂ is hydrogen, halo, hydroxy, amino, nitro, cyano, thio, alkyl,    alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,    cycloalkyl, heterocyloalkyl, cycloalkenyl, heterocycloakenyl,    heterocyclyl, alkoxyl, alkylamino, alkylthio, haloalkyl, sulphonyl,    sulphonamide, alkylsulphonamide, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   or R₁ and R₂ are taken together with the attached carbons to form an    optionally substituted cycle or heterocycle;-   R₃ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,    heteroalkynyl, cycloalkyl, heterocyloalkyl, cycloalkenyl,    heterocycloakenyl, heterocyclyl, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   R₄ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,    heteroalkynyl, cycloalkyl, heterocyloalkyl, cycloalkenyl,    heterocycloakenyl, heterocyclyl, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   or R₃ and R₄ are taken together with the attached carbons to form an    optionally substituted cycle or heterocycle; and-   R₅ is an alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,    heteroalkynyl, cycloalkyl, heterocyloalkyl, cycloalkenyl,    heterocycloakenyl, heterocyclyl, or an optionally substituted aryl,    arylalkyl, or arylalkenyl.

In some embodiments, this invention relates to compounds having ageneral formula (I), wherein R₁ is hydrogen, a C₁-C₁₂ alkyl, or anoptionally substituted C₃-C₁₂ cycloalkyl.

In some embodiments, this invention relates to compounds having ageneral formula (I), wherein R₂ is hydrogen, a C₁-C₁₂ alkyl, or anoptionally substituted C₃-C₁₂ cycloalkyl.

In some embodiments, this invention relates to compounds having ageneral formula (I), wherein R₁ and R₂ together with the attachedcarbons are linked to form an optionally substituted cycle.

In some embodiments, this invention relates to compounds having ageneral formula (I), wherein R₃ is a C₁-C₁₂ alkyl.

In some embodiments, this invention relates to compounds having ageneral formula (I), wherein R₄ is hydrogen.

In some other embodiments, this invention relates to compounds having ageneral formula (I), wherein R₅ is an optionally substituted C₄-C₁₂heterocycle, aryl, arylalkyl, or arylalkenyl.

In some other embodiments, this invention relates to a pharmaceuticalcomposition comprising one or more compounds disclosed herein, or apharmaceutically acceptable salt thereof, together with one or morediluents, excipients or carriers.

In some other embodiments, this invention relates to a pharmaceuticalcomposition comprising one or more compounds disclosed herein, or apharmaceutically acceptable salt thereof, in combination with one ormore other compounds by the same or different mode of action, togetherwith one or more diluents, excipients or carriers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts structure of membrane-bound adenylyl cyclases.

FIG. 2 shows structures of select adenine (NB001 and SQ22536) andnon-adenine (NKY80, ST034307, and ST072383) AC inhibitors.

FIG. 3 depicts selected analogs representing AC1 inhibitor scaffolds.

DETAILED DESCRIPTION

While the concepts of the present disclosure are illustrated anddescribed in detail in the figures and the description herein, resultsin the figures and their description are to be considered as exemplaryand not restrictive in character; it being understood that only theillustrative embodiments are shown and described and that all changesand modifications that come within the spirit of the disclosure aredesired to be protected.

As used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art.

In the present disclosure the term “about” can allow for a degree ofvariability in a value or range, for example, within 10%, within 5%, orwithin 1% of a stated value or of a stated limit of a range. In thepresent disclosure, the term “substantially” can allow for a degree ofvariability in a value or range, for example, within 90%, within 95%,99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more of a statedvalue or of a stated limit of a range.

The term “substituted” as used herein refers to a functional group inwhich one or more hydrogen atoms contained therein are replaced by oneor more non-hydrogen atoms. The term “functional group” or “substituent”as used herein refers to a group that can be or is substituted onto amolecule. Examples of substituents or functional groups include, but arenot limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom ingroups such as hydroxyl groups, alkoxy groups, aryloxy groups,aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups includingcarboxylic acids, carboxylates, and carboxylate esters; a sulfur atom ingroups such as thiol groups, alkyl and aryl sulfide groups, sulfoxidegroups, sulfone groups, sulfonyl groups, and sulfonamide groups; anitrogen atom in groups such as amines, azides, hydroxylamines, cyano,nitro groups, N-oxides, hydrazides, and enamines; and other heteroatomsin various other groups.

The term “alkyl” as used herein refers to substituted or unsubstitutedstraight chain and branched alkyl groups and cycloalkyl groups havingfrom 1 to about 20 carbon atoms (C₁-C₂₀), 1 to 12 carbons (C₁-C₁₂), 1 to8 carbon atoms (C₁-C₈), or, in some embodiments, from 1 to 6 carbonatoms (C₁-C₆). Examples of straight chain alkyl groups include thosewith from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl,n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branchedalkyl groups include, but are not limited to, isopropyl, iso-butyl,sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups.As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, andanteisoalkyl groups as well as other branched chain forms of alkyl.Representative substituted alkyl groups can be substituted one or moretimes with any of the groups listed herein, for example, amino, hydroxy,cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

The term “alkenyl” as used herein refers to substituted or unsubstitutedstraight chain and branched divalent alkenyl and cycloalkenyl groupshaving from 2 to 20 carbon atoms(C₂-C₂₀), 2 to 12 carbons (C₂-C₁₂), 2 to8 carbon atoms (C₂-C₈) or, in some embodiments, from 2 to 4 carbon atoms(C₂-C₄) and at least one carbon-carbon double bond. Examples of straightchain alkenyl groups include those with from 2 to 8 carbon atoms such as—CH═CH—, —CH═CHCH₂—, and the like. Examples of branched alkenyl groupsinclude, but are not limited to, —CH═C(CH₃)— and the like.

An alkynyl group is the fragment, containing an open point of attachmenton a carbon atom that would form if a hydrogen atom bonded to a triplybonded carbon is removed from the molecule of an alkyne. The term“hydroxyalkyl” as used herein refers to alkyl groups as defined hereinsubstituted with at least one hydroxyl (—OH) group.

The term “cycloalkyl” as used herein refers to substituted orunsubstituted cyclic alkyl groups such as, but not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl groups. In some embodiments, the cycloalkyl group can have 3to about 8-12 ring members, whereas in other embodiments the number ofring carbon atoms range from 3 to 4, 5, 6, or 7. In some embodiments,cycloalkyl groups can have 3 to 6 carbon atoms (C₃-C₆). Cycloalkylgroups further include polycyclic cycloalkyl groups such as, but notlimited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, andcarenyl groups, and fused rings such as, but not limited to, decalinyl,and the like.

The term “acyl” as used herein refers to a group containing a carbonylmoiety wherein the group is bonded via the carbonyl carbon atom. Thecarbonyl carbon atom is also bonded to another carbon atom, which can bepart of a substituted or unsubstituted alkyl, aryl, aralkyl cycloalkyl,cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl,heteroarylalkyl group or the like. In the special case wherein thecarbonyl carbon atom is bonded to a hydrogen, the group is a “formyl”group, an acyl group as the term is defined herein. An acyl group caninclude 0 to about 12-40, 6-10, 1-5 or 2-5 additional carbon atomsbonded to the carbonyl group. An acryloyl group is an example of an acylgroup. An acyl group can also include heteroatoms within the meaninghere. A nicotinoyl group (pyridyl-3-carbonyl) is an example of an acylgroup within the meaning herein. Other examples include acetyl, benzoyl,phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and thelike. When the group containing the carbon atom that is bonded to thecarbonyl carbon atom contains a halogen, the group is termed a“haloacyl” group. An example is a trifluoroacetyl group.

The term “aryl” as used herein refers to substituted or unsubstitutedcyclic aromatic hydrocarbons that do not contain heteroatoms in thering. Thus aryl groups include, but are not limited to, phenyl,azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl,triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl,anthracenyl, and naphthyl groups. In some embodiments, aryl groupscontain about 6 to about 14 carbons (C₆-C₁₄) or from 6 to 10 carbonatoms (C₆-C₁₀) in the ring portions of the groups. Aryl groups can beunsubstituted or substituted, as defined herein. Representativesubstituted aryl groups can be mono-substituted or substituted more thanonce, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substitutedphenyl or 2-8 substituted naphthyl groups, which can be substituted withcarbon or non-carbon groups such as those listed herein.

The term “aralkyl” and “arylalkyl” as used herein refers to alkyl groupsas defined herein in which a hydrogen or carbon bond of an alkyl groupis replaced with a bond to an aryl group as defined herein.Representative aralkyl groups include benzyl and phenylethyl groups andfused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenylgroups are alkenyl groups as defined herein in which a hydrogen orcarbon bond of an alkyl group is replaced with a bond to an aryl groupas defined herein.

The term “heterocyclyl” as used herein refers to substituted orunsubstituted aromatic and non-aromatic ring compounds containing 3 ormore ring members, of which, one or more is a heteroatom such as, butnot limited to, B, N, O, and S. Thus, a heterocyclyl can be acycloheteroalkyl, or a heteroaryl, or if polycyclic, any combinationthereof. In some embodiments, heterocyclyl groups include 3 to about 20ring members, whereas other such groups have 3 to about 15 ring members.In some embodiments, heterocyclyl groups include heterocyclyl groupsthat include 3 to 8 carbon atoms (C₃-C₈), 3 to 6 carbon atoms (C₃-C₆) or6 to 8 carbon atoms (C₆-C₈).

A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase“heterocyclyl group” includes fused ring species including those thatinclude fused aromatic and non-aromatic groups. Representativeheterocyclyl groups include, but are not limited to pyrrolidinyl,azetidinyl, piperidynyl, piperazinyl, morpholinyl, chromanyl,indolinonyl, isoindolinonyl, furanyl, pyrrolidinyl, pyridinyl,pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, tetrahydrofuranyl,pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl, triazyolyl, tetrazolyl,benzoxazolinyl, benzthiazolinyl, and benzimidazolinyl groups.

The term “heterocyclylalkyl” as used herein refers to alkyl groups asdefined herein in which a hydrogen or carbon bond of an alkyl group asdefined herein is replaced with a bond to a heterocyclyl group asdefined herein. Representative heterocyclylalkyl groups include, but arenot limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-ylmethyl, tetrahydrofuran-2-yl methyl, and indol-2-yl propyl.

The term “heteroarylalkyl” as used herein refers to alkyl groups asdefined herein in which a hydrogen or carbon bond of an alkyl group isreplaced with a bond to a heteroaryl group as defined herein.

The term “alkoxy” as used herein refers to an oxygen atom connected toan alkyl group, including a cycloalkyl group, as are defined herein.Examples of linear alkoxy groups include but are not limited to methoxy,ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples ofbranched alkoxy include but are not limited to isopropoxy, sec-butoxy,tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclicalkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy,cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can furtherinclude double or triple bonds, and can also include heteroatoms. Forexample, an allyloxy group is an alkoxy group within the meaning herein.A methoxyethoxy group is also an alkoxy group within the meaning herein,as is a methylenedioxy group in a context where two adjacent atoms of astructure are substituted therewith.

The term “amine” as used herein refers to primary, secondary, andtertiary amines having, e.g., the formula N(group)3 wherein each groupcan independently be H or non-H, such as alkyl, aryl, and the like.Amines include but are not limited to R—NH₂, for example, alkylamines,arylamines, alkylarylamines; R₂NH wherein each R is independentlyselected, such as dialkylamines, diarylamines, aralkylamines,heterocyclylamines and the like; and R₃N wherein each R is independentlyselected, such as trialkylamines, dialkylarylamines, alkyldiarylamines,triarylamines, and the like. The term “amine” also includes ammoniumions as used herein.

The term “amino group” as used herein refers to a substituent of theform —NH₂, —NHR, —NR₂, —NR₃ ⁺, wherein each R is independently selected,and protonated forms of each, except for —NR₃ ⁺, which cannot beprotonated. Accordingly, any compound substituted with an amino groupcan be viewed as an amine. An “amino group” within the meaning hereincan be a primary, secondary, tertiary, or quaternary amino group. An“alkylamino” group includes a monoalkylamino, dialkylamino, andtrialkylamino group.

The terms “halo,” “halogen,” or “halide” group, as used herein, bythemselves or as part of another substituent, mean, unless otherwisestated, a fluorine, chlorine, bromine, or iodine atom.

The term “haloalkyl” group, as used herein, includes mono-halo alkylgroups, poly-halo alkyl groups wherein all halo atoms can be the same ordifferent, and per-halo alkyl groups, wherein all hydrogen atoms arereplaced by halogen atoms, such as fluoro. Examples of haloalkyl includetrifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl,1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, —CF(CH₃)₂ and the like.

The term “optionally substituted,” or “optional substituents,” as usedherein, means that the groups in question are either unsubstituted orsubstituted with one or more of the substituents specified. When thegroups in question are substituted with more than one substituent, thesubstituents may be the same or different. When using the terms“independently,” “independently are,” and “independently selected from”mean that the groups in question may be the same or different. Certainof the herein defined terms may occur more than once in the structure,and upon such occurrence each term shall be defined independently of theother.

The compounds described herein may contain one or more chiral centers,or may otherwise be capable of existing as multiple stereoisomers. it isto be understood that in one embodiment, the invention described hereinis not limited to any particular stereochemical requirement, and thatthe compounds, and compositions, methods, uses, and medicaments thatinclude them may be optically pure, or may be any of a variety ofstereoisomeric mixtures, including racemic and other mixtures ofenantiomers, other mixtures of diastereomers, and the like. It is alsoto be understood that such mixtures of stereoisomers may include asingle stereochemical configuration at one or more chiral centers, whileincluding mixtures of stereochemical configuration at one or more otherchiral centers,

Similarly, the compounds described herein may include geometric centers,such as cis, trans, E, and Z double bonds. It is to be understood thatin another embodiment, the invention described herein is not limited toany particular geometric isomer requirement, and that the compounds, andcompositions, methods, uses, and medicaments that include them may bepure, or may be any of a variety of geometric isomer mixtures. It isalso to be understood that such mixtures of geometric isomers mayinclude a single configuration at one or more double bonds, whileincluding mixtures of geometry at one or more other double bonds.

As used herein, the term “salts” and “pharmaceutically acceptable salts”refer to derivatives of the disclosed compounds wherein the parentcompound is modified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic groups such as amines; and alkalior organic salts of acidic groups such as carboxylic acids.Pharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,and nitric; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, andisethionic, and the like.

Pharmaceutically acceptable salts can be synthesized from the parentcompound which contains a basic or acidic moiety by conventionalchemical methods. In some instances, such salts can be prepared byreacting the free acid or base forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two; generally, nonaqueous medialike ether, ethyl acetate, ethanol, isopropanol, or acetonitrile arepreferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, the disclosure of which is hereby incorporated by reference.

The term “solvate” means a compound, or a salt thereof, that furtherincludes a stoichiometric or non-stoichiometric amount of solvent boundby non-covalent intermolecular forces. Where the solvent is water, thesolvate is a hydrate.

Further, in each of the foregoing and following embodiments, it is to beunderstood that the formulae include and represent not only allpharmaceutically acceptable salts of the compounds, but also include anyand all hydrates and/or solvates of the compound formulae or saltsthereof. It is to be appreciated that certain functional groups, such asthe hydroxy, amino, and like groups form complexes and/or coordinationcompounds with water and/or various solvents, in the various physicalforms of the compounds. Accordingly, the above formulae are to beunderstood to include and represent those various hydrates and/orsolvates. In each of the foregoing and following embodiments, it is alsoto be understood that the formulae include and represent each possibleisomer, such as stereoisomers and geometric isomers, both individuallyand in any and all possible mixtures. In each of the foregoing andfollowing embodiments, it is also to be understood that the formulaeinclude and represent any and all crystalline forms, partiallycrystalline forms, and non-crystalline and/or amorphous forms of thecompounds.

The term “pharmaceutically acceptable carrier” is art-recognized andrefers to a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting anysubject composition or component thereof. Each carrier must be“acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient. Someexamples of materials which may serve as pharmaceutically acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

As used herein, the term “administering” includes all means ofintroducing the compounds and compositions described herein to thepatient, including, but are not limited to, oral (po), intravenous (iv),intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal,ocular, sublingual, vaginal, rectal, and the like. The compounds andcompositions described herein may be administered in unit dosage formsand/or formulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles.

Illustrative formats for oral administration include tablets, capsules,elixirs, syrups, and the like. Illustrative routes for parenteraladministration include intravenous, intraarterial, intraperitoneal,epidural, intraurethral, intrasternal, intramuscular and subcutaneous,as well as any other art recognized route of parenteral administration.

Illustrative means of parenteral administration include needle(including microneedle) injectors, needle-free injectors and infusiontechniques, as well as any other means of parenteral administrationrecognized in the art. Parenteral formulations are typically aqueoussolutions which may contain excipients such as salts, carbohydrates andbuffering agents (preferably at a pH in the range from about 3 to about9), but, for some applications, they may be more suitably formulated asa sterile non-aqueous solution or as a dried form to be used inconjunction with a suitable vehicle such as sterile, pyrogen-free water.The preparation of parenteral formulations under sterile conditions, forexample, by lyophilization, may readily be accomplished using standardpharmaceutical techniques well known to those skilled in the art.Parenteral administration of a compound is illustratively performed inthe form of saline solutions or with the compound incorporated intoliposomes. In cases where the compound in itself is not sufficientlysoluble to be dissolved, a solubilizer such as ethanol can be applied.

The dosage of each compound of the claimed combinations depends onseveral factors, including: the administration method, the condition tobe treated, the severity of the condition, whether the condition is tobe treated or prevented, and the age, weight, and health of the personto be treated. Additionally, pharmacogenomic (the effect of genotype onthe pharmacokinetic, pharmacodynamic or efficacy profile of atherapeutic) information about a particular patient may affect thedosage used.

It is to be understood that in the methods described herein, theindividual components of a co-administration, or combination can beadministered by any suitable means, contemporaneously, simultaneously,sequentially, separately or in a single pharmaceutical formulation.Where the co-administered compounds or compositions are administered inseparate dosage forms, the number of dosages administered per day foreach compound may be the same or different. The compounds orcompositions may be administered via the same or different routes ofadministration. The compounds or compositions may be administeredaccording to simultaneous or alternating regimens, at the same ordifferent times during the course of the therapy, concurrently individed or single forms.

The term “therapeutically effective amount” as used herein, refers tothat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated. In one aspect, the therapeuticallyeffective amount is that which may treat or alleviate the disease orsymptoms of the disease at a reasonable benefit/risk ratio applicable toany medical treatment. However, it is to be understood that the totaldaily usage of the compounds and compositions described herein may bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically-effective dose level for anyparticular patient will depend upon a variety of factors, including thedisorder being treated and the severity of the disorder; activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, gender and diet of the patient: the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidentally with the specific compound employed; andlike factors well known to the researcher, veterinarian, medical doctoror other clinician of ordinary skill.

Depending upon the route of administration, a wide range of permissibledosages are contemplated herein, including doses falling in the rangefrom about 1 μg/kg to about 1 g/kg. The dosages may be single ordivided, and may administered according to a wide variety of protocols,including q.d. (once a day), b.i.d. (twice a day), t.i.d. (three times aday), or even every other day, once a week, once a month, once aquarter, and the like. In each of these cases it is understood that thetherapeutically effective amounts described herein correspond to theinstance of administration, or alternatively to the total daily, weekly,month, or quarterly dose, as determined by the dosing protocol.

In addition to the illustrative dosages and dosing protocols describedherein, it is to be understood that an effective amount of any one or amixture of the compounds described herein can be determined by theattending diagnostician or physician by the use of known techniquesand/or by observing results obtained under analogous circumstances. Indetermining the effective amount or dose, a number of factors areconsidered by the attending diagnostician or physician, including, butnot limited to the species of mammal, including human, its size, age,and general health, the specific disease or disorder involved, thedegree of or involvement or the severity of the disease or disorder, theresponse of the individual patient, the particular compoundadministered, the mode of administration, the bioavailabilitycharacteristics of the preparation administered, the dose regimenselected, the use of concomitant medication, and other relevantcircumstances.

The term “patient” includes human and non-human animals such ascompanion animals (dogs and cats and the like) and livestock animals.Livestock animals are animals raised for food production. The patient tobe treated is preferably a mammal, in particular a human being.

In some illustrative embodiments, the invention relates to a compound offormula (I)

or a pharmaceutically acceptable salt thereof, wherein

-   R₁ is hydrogen, halo, hydroxy, amino, nitro, cyano, thio, alkyl,    alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,    cycloalkyl, heterocyloalkyl, cycloalkenyl, heterocycloakenyl,    heterocyclyl, alkoxyl, alkylamino, alkylthio, haloalkyl, sulphonyl,    sulphonamide, alkylsulphonamide, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   R₂ is hydrogen, halo, hydroxy, amino, nitro, cyano, thio, alkyl,    alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,    cycloalkyl, heterocyloalkyl, cycloalkenyl, heterocycloakenyl,    heterocyclyl, alkoxyl, alkylamino, alkylthio, haloalkyl, sulphonyl,    sulphonamide, alkylsulphonamide, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   or R₁ and R₂ are taken together with the attached carbons to form an    optionally substituted cycle or heterocycle;-   R₃ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,    heteroalkynyl, cycloalkyl, heterocyloalkyl, cycloalkenyl,    heterocycloakenyl, heterocyclyl, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   R₄ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,    heteroalkynyl, cycloalkyl, heterocyloalkyl, cycloalkenyl,    heterocycloakenyl, heterocyclyl, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   or R₃ and R₄ are taken together with the attached carbons to form an    optionally substituted cycle or heterocycle;-   and-   R₅ is an alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,    heteroalkynyl, cycloalkyl, heterocyloalkyl, cycloalkenyl,    heterocycloakenyl, heterocyclyl, or an optionally substituted aryl,    arylalkyl, or arylalkenyl.

In some other embodiments, this invention relates to compounds having ageneral formula (I), wherein R₁ is hydrogen, a C₁-C₁₂ alkyl, or anoptionally substituted C₃-C₁₂ cycloalkyl.

In some embodiments, this invention relates to compounds having ageneral formula (I), wherein R₂ is hydrogen, a C₁-C₁₂ alkyl, or anoptionally substituted C₃-C₁₂ cycloalkyl.

In some embodiments, this invention relates to compounds having ageneral formula (I), wherein R₁ and R₂ together with the attachedcarbons are linked to form an optionally substituted cycle.

In some embodiments, this invention relates to compounds having ageneral formula (I), wherein R₃ is a C₁-C₁₂ alkyl.

In some embodiments, this invention relates to compounds having ageneral formula (I), wherein R₄ is hydrogen.

In some other embodiments, this invention relates to compounds having ageneral formula (I), wherein R₅ is an optionally substituted C₄-C₁₂heterocycle, aryl, arylalkyl, or arylalkenyl.

In some embodiments, this invention relates to compounds selected fromthe group consisting of

In some other embodiments, this invention relates to a pharmaceuticalcomposition comprising one or more compounds disclosed herein, or apharmaceutically acceptable salt thereof, together with one or morediluents, excipients or carriers.

In some other embodiments, this invention relates to a pharmaceuticalcomposition comprising one or more compounds disclosed herein, or apharmaceutically acceptable salt thereof, in combination with one ormore other compounds by the same or different mode of action, togetherwith one or more diluents, excipients or carriers.

In some embodiments, this invention relates to a method of treatingpain, opioid dependence, alcohol use disorder, or autism comprising thestep of administering to a mammal in need of relief from said pain oropioid dependence thereof a therapeutically effective amount of one ormore compounds of a mammal in need thereof a therapeutically effectiveamount of one or more compounds of formula (I)

or a pharmaceutically acceptable salt thereof, and one or more carriers,diluents, or excipients, wherein

-   R₁ is hydrogen, halo, hydroxy, amino, nitro, cyano, thio, alkyl,    alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,    cycloalkyl, heterocyloalkyl, cycloalkenyl, heterocycloakenyl,    heterocyclyl, alkoxyl, alkylamino, alkylthio, haloalkyl, sulphonyl,    sulphonamide, alkylsulphonamide, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   R₂ is hydrogen, halo, hydroxy, amino, nitro, cyano, thio, alkyl,    alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,    cycloalkyl, heterocyloalkyl, cycloalkenyl, heterocycloakenyl,    heterocyclyl, alkoxyl, alkylamino, alkylthio, haloalkyl, sulphonyl,    sulphonamide, alkylsulphonamide, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   or R₁ and R₂ are taken together with the attached carbons to form an    optionally substituted cycle or heterocycle;-   R₃ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,    heteroalkynyl, cycloalkyl, heterocyloalkyl, cycloalkenyl,    heterocycloakenyl, heterocyclyl, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   R₄ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,    heteroalkynyl, cycloalkyl, heterocyloalkyl, cycloalkenyl,    heterocycloakenyl, heterocyclyl, or an optionally substituted aryl,    arylalkyl, or arylalkenyl;-   or R₃ and R₄ are taken together with the attached carbons to form an    optionally substituted cycle or heterocycle;

and

-   R₅ is an alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,    heteroalkynyl, cycloalkyl, heterocyloalkyl, cycloalkenyl,    heterocycloakenyl, heterocyclyl, or an optionally substituted aryl,    arylalkyl, or arylalkenyl.

In some other embodiments, this invention relates to a method fortreating a chronic pain.

In some other embodiments, this invention is related to a method fortreating and reducing pain and opioid dependence.

In some other embodiments, this invention is related to a method fortreating and reducing pain and opioid dependence further comprising thestep of administering a compound of formula I in combination with anopioid drug, wherein the compound of formula I enhances μ-opioidreceptor inhibition of adenylyl cyclase 1.

In some other embodiments, this invention is related to a method fortreating and reducing pain and opioid dependence, wherein the opioiddrug is selected from the group consisting of codeine, morphine,thebaine, oripavine, diacetylmorphine, nicomorphine,dipropanoylmorphine, diacetyldihydromorphine, acetylpropionylmorphine,desomorphine, methyldesorphine, dibenzoylmorphine, dihydrocodeine,ethylmorphine, heterocodeine, buprenorphine, etorphine, hydrocodone,hydromorphone, oxycodone, oxymorphone, fentanyl, alphamethylfentanyl,alfentanil, sufentanil, remifentanil, carfentanyl, ohmefentanyl,pethidine (meperidine), ketobemidone, desmethylprodine (MPPP),allylprodine, prodine, phenethylphenylacetoxypiperidine (PEPAP),promedol, propoxyphene, dextropropoxyphene, dextromoramide, bezitramide,piritramide, methadone, dipipanone, levomethadyl acetate (LAAM),difenoxin, diphenoxylate, loperamide, dezocine, pentazocine,phenazocine, buprenorphine, dihydroetorphine, etorphine, butorphanol,nalbuphine, levorphanol, levomethorphan, lefetamine, menthol,meptazinol, mitragynine, tilidine, tramadol, tapentadol, eluxadoline,nalmefene, naloxone, and naltrexone.

The following non-limiting exemplary embodiments are included herein tofurther illustrate the invention. These exemplary embodiments are notintended and should not be interpreted to limit the scope of theinvention in any way. It is also to be understood that numerousvariations of these exemplary embodiments are contemplated herein.

Adenylyl Cyclase Inhibitors as Potential Drugs.

The observations described above led Dr. Zhuo and colleagues to identifyand subsequently evaluate a small molecule inhibitor of AC1, NB001 (FIG.2) in multiple chronic pain models (Kang et al., (2016) Mol Pain 12.;Tian et al., (2015) Mol Brain 8:60.). This molecule was identified froma search of forskolin- and adenosine triphosphate-like structures. NB001was shown to have >10-fold selectivity for AC1 (IC50=10 μM) versus theclosely related AC8 (IC50=139 uM) in HEK293 cells expressing recombinantAC isoforms. NB001 was shown to have activity consistent with AC1inhibition in additional neuronal cell models (i.e. SH-SY5Y cells) andtissue preparations of the anterior cingulate cortex. Consistent withdata from the AC1^(−/−) mice, NB001 markedly attenuated chronic painresponses (i.e. inflammatory and neuropathic) in both mice and rats.More recent studies also suggest that NB001 has AC1 inhibitory activityin animal models of opiate dependence (Corder et al., (2013) Science341:1394-1399.). These preclinical studies with NB001 implicate furtherAC1 as a potential new target for exploring inhibitors of chronic pain.

The development and mechanistic characterization of AC1-selective smallmolecule inhibitors is believed to provide novel non-opioid weapons inthe war on chronic pain. We identified our initial hits via screening10,000 selected compounds from the Life Chemicals collection. The screenwas carried out using HEK cells stably expressing AC1 (HEK-AC1) andcyclic AMP accumulation was stimulated using the Ca²⁺ ionophore, A23187to selectively activate AC1. Compounds with potential chemicalliabilities and PAINs were removed from further evaluation (Baell andHolloway, J. Med. Chem. 2010, 53, 2719-2740).

Fresh powders of several robust inhibitors (>90% inhibition) wereevaluated in multiple confirmation assays assessing the dose responserelationship for inhibiting AC1 and AC8 activity. Confirmation assaysused A23817 to selectively stimulate recombinant AC1 or AC8 in the HEKcell background (Cumbay and Watts (2001), J. Pharmacol Exp Ther297:1201-1399). This approach takes advantage of one unique regulatoryproperty of AC1 for the development of selective inhibitors. Both AC1and AC8 are robustly activated by Ca²⁺/CaM under a variety ofconditions, whereas the effects of Ca²⁺/CaM on AC3 are modest andconditional requiring activation by G proteins. Previous studies haveidentified unique Ca²⁺/CaM binding domains as one possible site ofinteraction to achieve AC1 (vs. AC8) selectivity (Masada et al., 2012,Biochemistry 51:7917-7929). These observations suggest that AC1selectivity can be achieved through targeting Ca²⁺/CaM activation ofAC1. In support of this, a portion of the hits identified for expansionherein revealed selective inhibition of Ca²⁺/CaM-stimulated AC1 versusAC8 (Table 1).

The primary technology we employed to measure cAMP accumulation was ahomogenous time-resolved fluorescence (HTRF) assay, the same technologyutilized in the primary screen. The activity of the new compounds wasstudied at AC1 and AC8 revealing a range of potency and activitypatterns (Table 1 and FIG. 3 and data not shown). Subsequent assays forAC1 and AC8 using the HitHunter cAMP assay revealed similar rank-orderpotency and efficacy values (data not shown). The most potent andselective compounds F2215-0213 (FIG. 3 and Table 1) appears to be atleast 60 fold selective for AC1 versus AC8.

TABLE 1 Activity at AC1, AC8, AC2, and AC5 in stable HEK cell lines*hAC8^(c) hAC1 IC₅₀ Selectivity^(d) hAC2^(c) hAC5^(c) IC₅₀ μM % μM % AC1vs % % Viability^(e) Compound μμ(μM) Inhib (μM) Inhib AC8^(d) InhibInhib % Veh ST034307^(a) 2.3 108 ± 10 ND −25 ± 1  >13-fold −142 −44 85 ±6 F0608-0538^(b) 2.1 ± 0.4 101 ± 1  21 ± 4 76 ± 8  10-fold −95 −34 118 ±6  F0608-0011^(b) 2.0 ± 0.2 101 ± 1  18 ± 2 79 ± 1  9-fold −7 −10 125 ±3  F2289-0107^(b) 7.5 ± 2.0 99 ± 1 ND 31 ± 2  >4-fold −210 −28 117 ± 1 F0559-0346^(b) 9.1 ± 2.0 99 ± 1 17 ± 6  74 ± 13  2-fold −6 4 118 ± 2 F2215-0213^(b) 0.5 ± 0.1 99 ± 1 ND 20 ± 1 >60-fold −70 9 101 ± 17F2215-0772^(b) 0.9 ± 0.2 100 ± 1  ND 34 ± 7 >30-fold 32 13 93 ± 1F2215-0954^(b) 5.9 ± 0.4 99 ± 1 ND 35 ± 8  >5-fold −185 −21 106 ± 14*Cyclic AMP accumulation for AC1 or AC8 was stimulated using A23187. AC2activity was stimulated with 100 nM PMA and AC5 activity was stimulatedwith 300 nM forskolin. ND = not able to be determined. ^(a)FIG. 2 anddata from Brust et al., Sci. Signal. 21 Feb. 2017: Vol. 10, Issue 467,eaah5381); ^(b)FIG. 3; ^(c)% inhibition at 30 μM (negative valuesindicate a potentiated response); ^(d)Selectivity = AC8 IC₅₀/AC1 IC₅₀,in the absence of IC₅₀ values at AC8, estimates based on IC₅₀ value of≥30 μM; ^(e)values determined in AC1-HEK cells. All values based n ≥ 3,except AC2 and AC5 (n = 2).

TABLE 2 Activity Table of Compounds Disclosed HEK-AC1 HEK-AC8 HEK-AC2Compd. IC50 % Inhibition IC50 % Inhibition % Inhibition % Inhibition ID(μM) (30 μM) (μM) (30 μM) (10 μM) (30 μM) AC10048 0.58 ± 0.2  99 ± 2 N/D*  19 ± 11 (−)186 ± 20   (−)59 ± 12 AC10049 1.3 ± 0.2 98 ± 1 68 ± 1026 ± 4 (−)177 ± 31 (−) 128 ± 7  AC10058 1.2 ± 0.2 99 ± 1 N/D 19 ± 4(−)226 ± 14 (−) 190 ± 21 AC10042 1.5 ± 0.3  101 ± 0.7 N/D  9 ± 4 (−)179± 50   (−)94 ± 35 AC10057 3.7 ± 0.2 94 ± 1 N/D  9 ± 2 (−)195 ± 26 (−)199 ± 30 AC10043 10.3 ± 2   92 ± 2 N/D 16 ± 9 (−) 109 ± 36  (−)196 ±8  AC10050 16.7 ± 1   79 ± 4 N/D (−) 1 ± 10   (−)21 ± 10 (−) 126 ± 43AC10051 N/D  4 ± 7 N/D (−) 3 ± 11   (−)13 ± 12  (−) 12 ± 11 *N/D: notdetermined

Experimental Methods

Compounds and Other Chemicals Used.

Forskolin and phorbol 12-myristate 13-acetate (PMA) were purchased fromTocris (Ellisville, Mo.). 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (HEPES), and ethylenediaminetetraacetic acid (EDTA) were purchasedfrom Fisher Scientific (Pittsburg, Pa.). NKY80 was purchased from EMDMillipore (Temecula, Calif.). Isoproterenol, A23187, adenosinemonophosphate (ATP),ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),3-isobutyl-1-methylxanthine (IBMX), 5′-guanylyl-imidodiphosphoate(GppNHp), TWEEN 20, MgCl₂, and 2-amino-2-(hydroxymethyl)-1,3-propanediol(TRIS) were purchased from Sigma-Aldrich (St. Louis, Mo.).2-Bromo-1-(1-phenyl-4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)ethanone(W400), 6-chloro-2-(trichloromethyl)-4H-chromen-4-one (ST034307), and4-chloro-1-methyl-3-nitroquinolin-2(1H)-one (ST072383) were purchasedfrom TimTec (Newark, Del.).

Cell Culture.

Human embryonic kidney (HEK) cells stably expressing AC1, AC8, or AC1with the MOR were cultured in Dulbecco's Modified Eagle Medium (LifeTechnologies, Grand Island, N.Y.) supplemented with 5% bovine calf serum(Hyclone, Logan, Utah), 5% fetal clone I (Hyclone),Antibiotic-Antimycotic (Life Technologies), and G418 (Invivogen, SanDiego, Calif.) (HEK-AC1), or hygromycin B (Fisher Scientific, Pittsburg,Pa.) (HEK-AC8), or G418 and puromycin (Sigma-Aldrich) (HEK-AC1/MOR).Chinese hamster ovary (CHO) cells expressing the MOR (CHO-MOR) in thePathHunter® β-Arrestin GPCR assay platform were purchased from DiscoveRx(Freemont, Calif.). Cells were grown in Ham's F12 media supplementedwith 1 mM L-glutamine (Thermo Scientific, West Palm Beach, Fla.), 10%fetal bovine serum (Hyclone), 50 U/ml penicillin, 50 m/ml streptomycin(Life Technologies), G418 and hygromycin B. Cells were grown and frozenas previously described (J. M. Conley, et al., J Vis Exp, e51218(2014)).

Transient Transfections.

HEK cells were plated in 15 cm dishes at a confluence of 9.0×10⁶cells/dish and incubated at 37° C. in a humidified incubator overnight.On the following day, a 6 ml solution containing 9 μg of AC plasmid orvenus fluorescent protein (venus) control plasmid, and 60 μLlipofectamine 2000 (Life Technologies) in optiMEM (Life Technologies)was prepared and incubated at room temperature for 45 min. The solutionwas added dropwise to the cells, and transfection was carried out for 48h. Cells were harvested, and cryopreserved as described above. For AC7and AC9, HEK cells were plated in 10 cm dishes (at confluences of3.0×10⁶ or 3.5×10⁶ cells/dish, respectively) and incubated at 37° C. ina humidified incubator overnight. On the following day, a 3 mL solutioncontaining AC7 plasmid (10 μg), AC9 plasmid (3 μg), or venus plasmid;plus Gas plasmid (0.5 μg for AC7 and 0.3 μg for AC9) or venus plasmid;and Lipofectamine 2000 (48 μL for AC7 and 24 μl for AC9) in optiMEM wasprepared and incubated at room temperature for 45 min. The solution wasadded dropwise to the cells, transfection was carried out for 48 h, andcells were harvested and cryopreserved.

Cyclic AMP Accumulation in Cells.

Cyclic AMP accumulation was measured as previously described (Brust, etal., J. Pharmacol. Exp. Ther. 352, 480-493(2015)). Briefly,cryopreserved cells were thawed, resuspended in optiMEM (LifeTechnologies), and plated in white, flat bottom, low-volume, tissueculture-treated 384 well plates (PerkinElmer, Shelton, Conn.). Plateswith cells were incubated in a 37° C. humidified incubator for 1 h.Inhibitors were added and plates were incubated at room temperature for30 min followed by the addition of AC stimulants in the presence of 500μM IBMX. Cells were incubated at room temperature for 1 h and cAMPaccumulation was measured using Cisbio's cAMP kit (Cisbio Bioassays,Bedford, Mass.) according to the manufacturer's instructions.

Additional assays measured cAMP accumulation using the HitHunter® cAMPAssay Platform from DiscoveRx according to the manufacturer'sinstructions. Luminescence (HitHunter® cAMP Assay) and fluorescence(Cisbio's dynamic 2 kit) counts were measured using a Synergy 4 (BioTek,Winooski, Vt.).

Compound Screening.

Cryopreserved HEK-AC1 cells were thawed, washed, resuspended in optiMEMand plated into white, flat bottom, tissue culture-treated 384-wellplates (PerkinElmer) at 15 μL/well using a MultiFlo dispenser (BioTek).Cells were incubated in a 37° C. humidified incubator for 1 h. Next,test compounds (3.5 mg/l final assay concentration) from the NDL-3000Natural Derivatives library (TimTec) were added (70 nL/well) using aMultiPette-mounted 384 well pin tool and incubated at room temperaturefor 30 min. Following the incubation with test compounds, 5 μL/well of 3μM A23187 in the presence of 30 nM forskolin and 500 μM IBMX (finalconcentrations) was added to the cells using a MultiFlo dispenser. Cellswere incubated at room temperature for 1 h and cAMP accumulation wasmeasured as described above using a MultiFlo dispenser to sequentiallyadd 10 μL/well of cAMP-d2 and anti-cAMP cryptate conjugate workingsolutions (Cisbio Bioassays) to the cells. Test compounds were screenedin singlet and a Z′ factor of 0.55±0.22 (n=10) was obtained using 30 μMW400 as a positive control (J. M. Conley, et al., J. Pharmacol. Exp.Ther. 347, 276-287 (2013); J.-H. Zhang, J. Biomol. Screen. 4, 67-73(1999).

Cell Viability Assays.

Cell viability assays were conducted with HEK-AC1 cells followingplating and compound incubation protocols identical to the proceduresdescribed above in “Cyclic AMP assays in cells”. Cell viability wasmeasured as a percentage of vehicle using 2% Triton X-100(Sigma-Aldrich) as a control. The CellTiter-Glo® Luminescent CellViability Assay kit from Promega (Madison, Wis.) was employed to assesscell viability according to the manufacturer's instructions.Luminescence counts were measured using a Synergy 4.

Cyclic AMP Accumulation in Cellular Membranes from HEK Cells.

Cellular membranes from HEK-AC1 cells were isolated and frozen aspreviously described in the presence of 1 mM EGTA (T. F. Brust, et al.,J. Pharmacol. Exp. Ther. 352, 480-493 (2015). On the assay day membraneswere thawed on ice and resuspended in membrane buffer (33 mM HEPES, 0.1%TWEEN 20, 1 mM EGTA, pH 7.4). Protein concentration was measured usingthe Pierce BCA Protein Assay kit (Thermo Scientific) and 2.0-3.5 μg/wellwas plated in a white, flat bottom, tissue culture-treated 384 wellplate. Inhibitors (diluted in a 33 mM HEPES, 0.1% TWEEN 20 solution)were added and incubated for 20 min at room temperature. Next, 3 μMcalmodulin or 30 μM forskolin (final concentrations) was added instimulation buffer (33 mM HEPES, 0.1% TWEEN 20, 1.5 mM MgCl₂, 250 μMATP, 1 μM GppNHp, 500 μM IBMX, and 500 μM CaCl₂-10 μM free Ca²⁺) andincubated at room temperature for 45 min. Cyclic AMP accumulation wasmeasured using Cisbio's dynamic 2 kit according to the manufacturer'sinstructions.

Adenylyl Cyclase Assays in Cellular Membranes from Sf9 Cells.

Membranes from Sf9 cells expressing AC1, AC2, or AC5 were prepared aspreviously described (C. W. Dessauer, Methods Enzymol. 345, 112-126(2002)). All activity assays were performed for 10 min at 30° C. in afinal volume of 50 μL. The final concentration of MgCl₂ and Mg-ATP inthe reaction was 10 mM and 200 μM, respectively. AC-containing membranes(10-20 μg) were premixed with Gas (50 nM final). Inhibitors weresolubilized in DMSO and incubated with AC-containing membranes for 10min on ice before the start of the reaction. The final concentration ofDMSO in the reaction did not exceed 3% for either vehicle or inhibitors.Reactions were initiated upon addition of a reaction mix containing[α-³²P]ATP. The reactions were terminated with stop solution (2.5% SDS,50 mM ATP, and 1.75 mM cAMP) and the products were separated bysequential chromatography on Dowex-50 and Al₂O₃.

Assays with Hippocampal Homogenates.

C57BL/6 mice (13 weeks old) were decapitated, their brains were quicklyremoved, and 2-mm slices encompassing the hippocampus were collected onice. The hippocampal region was dissected and immediately frozen in a−80° C. freezer, where they were stored until the assay day. Dissectedhippocampal tissue was thawed on ice, weighed, and homogenized inmembrane buffer (2 mL/mg—wet weight) with ten manual strokes using aWheaton-Teflon glass homogenizer. Homogenates were added to a white,flat bottom, tissue culture-treated 384-well plate and inhibitors(diluted in a 33 mM HEPES, 0.1% TWEEN 20 solution) were added andincubated for 20 min at room temperature. Next, 3 μM calmodulin (finalconcentration) was added in stimulation buffer (same as assays incellular membranes from HEK cells) and incubated at room temperature for45 min. Cyclic AMP accumulation was measured using Cisbio's dynamic 2kit according to the manufacturer's instructions.

β-Arrestin Recruitment Assay.

Recruitment of β-arrestin 2 to the MOR was measured as previouslydescribed (T. F. Brust, et al., J. Pharmacol. Exp. Ther. 352, 480-493(2015)). Briefly, CHO-MOR cells were plated in white, flat bottom,low-volume, tissue culture-treated 384-well plates. Plates with cellswere incubated in a 37° C. humidified incubator overnight. Following theincubation, AC1 inhibitors or vehicle was added to the cells, which wereincubated at room temperature for 30 min. Next, DAMGO or vehicle wasadded to cells, which were then incubated in a 37° C. humidifiedincubator for 1.5 h. β-arrestin 2 recruitment to the MOR was assessedusing the PathHunter® assay (DiscoveRx) according to the manufacturer'sinstructions. Luminescence counts were measured using a Synergy 4.

Heterologous Sensitization Assays.

Heterologous sensitization assays were conducted as previously described(T. F. Brust, et al., Biochem. Pharmacol. 93, 85-91 (2015)). Briefly,HEK-AC1/MOR cells were thawed and plated in white, flat bottom, tissueculture-treated 384-well plates. Plates with cells were incubated in a37° C. humidified incubator for 1 h. For inhibition of the developmentof sensitization, inhibitors were added and plates were incubated atroom temperature for 30 min, followed by addition of DAMGO andincubation at 37° C. for 2 h (to achieve sensitization). For the assaysto measure inhibition of the expression of sensitization the order ofDAMGO and ST034307 additions were reversed (i.e., DAMGO sensitizationbefore AC1 inhibition). Next, cells were treated with 3 μM A23187 in thepresence of 500 μM IBMX and 1 μM naloxone (final concentrations) andincubated at room temperature for 1 h. Cyclic AMP accumulation wasmeasured using Cisbio's dynamic 2 kit according to the manufacturer'sinstructions.

Animals and Housing.

Wild-type C57BL/6 mice, were obtained from Taconic (Cambridge City,Ind.). Male mice age 5 weeks (18-23 gr) were grouped and housed insingle grommet ventilated plexiglass cages at ambient temperature (21°C.) in a room maintained on a reversed 12L:12D cycle (lights off at10:00, lights on at 22:00) in our animal facility that has Associationfor Assessment and Accreditation of Laboratory Animal Care approval.Food and water were provided ad libitum. The mice were given ˜7 days toacclimatize to the housing conditions and reverse light cycle before thestart of the experiments. Mice were then habituated to the containmentboxes for the Von Frey assay. All animal procedures were pre-approved byour Institutional Animal Care and Use Committee and were in accordancewith the National Institutes of Health Guide for the Care and Use ofLaboratory Animals. Mice were not deprived of food or water at any time.

Inflammatory Pain Behavioral Assays.

C57BL/6 mice were placed in suspended rectangular plastic chambers on awire mesh grid to habituate for 1 h. Next, a baseline measurement ofmechanical sensitivity to Von Frey filaments was performed as previouslydescribed (G. Corder, et al., Science 341, 1394-1399 (2013); R. M. vanRijn, et al., Biol. Psychiatry 71, 232-238 (2012)). Immediately afterbaseline measurements, the mice were injected with Complete Freund'sadjuvant (CFA −10 μL, non-diluted) into the intraplantar surface of theleft hindpaw to induce inflammation. On the following day, inflammatoryhypersensitivity was measured using Von Frey filaments. Next, drugs wereinjected intrathecally as previously described (T. F. Brust, et al.,Biochem. Pharmacol. 93, 85-91 (2015); R. M. van Rijn, et al., Biol.Psychiatry 71, 232-238 (2012)). Drug-induced analgesia was measured 10min after intrathecal injections using Von Frey filaments. Data arerepresented as a percentage of the average baseline response.

Data and Statistical Analyses.

All data and statistical analyses were carried out using GraphPad Prism6 (GraphPad Software, San Diego, Calif.). Statistical analyses (one-wayANOVA or t tests) are described in text or figure legends whereappropriate.

Synthetic Chemistry.

The purity of all final compounds was >95% purity as assessed by HPLCaccording to current American Chemical Society guidelines forpublication. Final compounds were analyzed on an Agilent 1200 serieschromatograph. The chromatographic method utilized as Thermo ScientificHypersil GOLD C-18 4.6×250 mm, 3 μm column. UV detection wavelength=220nm; flow-rate=1.0 mL/min; gradient=5-95% acetonitrile over 12 min and 3min hold time at 95% acetonitrile. Both organic and aqueous mobilephases contain 0.1% v/v formic acid. The mass spectrometer used is an ABSciex 4500 QTrap triple-quadrupole mass spectrometer with an ESI source.Samples are submitted for analysis solubilized in 1:1 acetonitrile:watersolution. ¹H and ¹³C NMR spectra were recorded on either Bruker DRX500spectrometer (operating at 500 and 125 MHz, respectively) or BrukerAVIII (operating at 800 and 200 MHz, respectively) in DMSO-d₆ or CDCl₃with or without the internal standard of TMS at 0.05% v/v. The chemicalshifts (6) reported as parts per million (ppm) and the couplingconstants are reported as s=singlet, bs=broad singlet, d=doublet,t=triplet, q=quartet, dd=doublet of doublet, m=multiplet. Compounds weregenerally prepared according to Scheme 1. For non-commercially availablebuilding blocks, the synthesis procedure follows Scheme 2. FinalCompounds 9-16 were prepared according to general Scheme 3 and protocolsare detailed below.

The following procedure was carried out for all analogs and is detailedfor compound 9 (AC10042, JK2-AC1-012). All other analogs followed thisprotocol unless otherwise noted.

2-Fluoro-N-(3-methyl-1-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)-1H-pyrazol-5-yl)benzamide

Step 1: To a vial was added ethyl 3-oxobutanoate (1 g, 8.0 mmol, 1 eq.),thiourea (0.6, 8.0 mmol, 1 eq.) and KOH (0.4 g, 8.0 mmol, 1 eq.) inethanol (6 ml). The reaction mixture was stirred at 80° C. for 5 h.Ethanol was removed under reduced pressure to one third of its originalvolume, the reaction mixture was then poured into water and neutralizedwith 2 N HCl. The resulting mixture was left stirring overnight at rt. Aprecipitate formed and was filtered by vacuum filtration to provide thedesired product 6-methyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (1.0 g,7.033 mmol, 91%) as white solid. ESI-MS: 143.2 [M+H]⁺.

Step 2: To a vial was added6-methyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (1.5 g, 11 mmol, 1 eq.)and H2O. After that NaOH (0.46 g, 12 mmol, 1.1 eq.) was added portionwise to the vial containing suspension of6-methyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (1.5 g, 11 mmol, 1 eq.)in H₂O. The reaction mixture was stirred for 30 min and then it wascooled to 10° C., followed by the addition of methyl iodide (1.8 g, 13mmol, 1.2 eq). The mixture was stirred for another 24 h at roomtemperature, followed by again cooling to 10° C. The precipitate formedwas filtered by vacuum filtration, washed with H₂O and dried to providedesired product 6-methyl-2-(methylthio)pyrimidin-4(3H)-one (0.893 g,5.72 mmol, 54%) as white solid. APCI-MS: 157.1 [M+H]⁺.

Step 3: A mixture of 6-methyl-2-(methylthio)pyrimidin-4(3H)-one (0.934g, 5.98 mmol, 1 eq.), hydrazine hydrate (0.958 g, 29.9 mmol, 5 eq.) andK₂CO₃ (8.26 mg, 59.8 mol, 0.01 eq.) was refluxed in 2-propanol (0.359 g,5.98 mmol, 1 eq.) for 4 hours and then the reaction mixture was stirredat 60° C. overnight. The precipitates formed were filtered by vacuumfiltration and further washed with methanol, diethyl ether and air driedto get product(Z)-2-hydrazineylidene-6-methyl-2,3-dihydropyrimidin-4(1H)-one (0.176 g,1.26 mmol, 21.0%). ES-MS: 151.1 [M+H]⁺.

Step 4: (Z)-3-aminobut-2-enenitrile (0.246 g, 3.00 mmol, 1.8 eq.) wasadded portion wise to the suspension of(Z)-2-hydrazineylidene-6-methyl-2,3-dihydropyrimidin-4(1H)-one (0.23 g,1.6 mmol, 1 eq.) in ethanol at rt. The reaction mixture was refluxed for4 h, was then concentrated in vacuum and was further purified bynormal-phase flash chromatography (5% MeOH:DCM) to get desired product2-(5-amino-3-methyl-1H-pyrazol-1-yl)-6-methylpyrimidin-4(1H)-one (0.2 g,1 mmol, 61%). ESI-MS: 206.1 [M+H]⁺.

Step 5: (procedure includes converting carboxylic acid to acyl chloride.If the acyl chloride is commercially available then the oxalyl chloridestep can be bypassed): To a vial was added the 2-fluorobenzoic acid(0.038 g, 0.27 mmol, 1.1 eq.) in DCM (0.5 mL) followed by 1 drop of DMF.The reaction was then cooled to 0° C. followed by slow addition ofoxalyl chloride (0.025 mL, 0.28 mmol, 1.15 eq.). The reaction then wasallowed to warm to rt and stir for 5 hours. After 5 hours the reactionwas concentrated in vacuo and carried into the amide coupling reactionwithout purification or characterization.

To the second vial was added2-(5-amino-3-methyl-1H-pyrazol-1-yl)-6-methylpyrimidin-4(1H)-one (0.05g, 0.24 mmol, 1 eq.) in DCM (0.5 mL), followed by pyridine (0.023 g,0.29 mmol, 1.2 eq.). The acyl chloride was dissolved in DCM (0.3 mL) andthen added dropwise to the reaction mixture. The reaction mixture wasthen worked up using DCM and washed with 2N HCl, followed by brine andwas further purified by reverse-phase flash chromatography (5-100%MeCN:water) to afford desired product2-fluoro-N-(3-methyl-1-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)-1H-pyrazol-5-yl)benzamide(0.012 g, 0.037 mmol, 15%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ12.75 (s, 1H), 8.07-8.04 (m, 1H), 7.77-7.73 (m, 1H), 7.53-7.45 (m, 2H),6.90 (s, 1H), 6.33 (s, 1H), 5.10 (s, 1H), 2.37 (s, 3H), 2.30 (s, 3H).ESI-MS: 328.1[M+H]⁺. HPLC retention time: 12.247 min. HPLC purity 100%.

N-(3-methyl-1-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)-1H-pyrazol-5-yl)furan-2-carboxamide(10, AC10043) Isolated as a white solid (0.010 g, 0.033 mmol, 23%). ¹HNMR (500 MHz, DMSO-d₆) δ 13.37 (s, 1H), 7.99 (s, 1H), 7.34 (s, 1H), 6.74(s, 1H), 6.64 (s, 1H), 6.17 (s, 1H), 2.36 (s, 3H), 2.20 (s, 3H). ESI-MS:300.1 [M+H]⁺. HPLC retention time: 11.547 min. HPLC purity 95.6%.

N-(1-(6-ethyl-4-oxo-1,4-dihydropyrimidin-2-yl)-3-methyl-1H-pyrazol-5-yl)-2-fluorobenzamide(11, AC10048) Isolated as a white solid (0.012 g, 0.035 mmol, 15%). ¹HNMR (500 MHz, DMSO-d₆) δ 12.59 (s, 1H), 8.00-7.96 (m, 1H), 7.73-7.68 (m,1H), 7.50-7.41 (m, 2H), 6.86 (s, 1H), 6.28 (s, 1H), 2.60 (q, J=8.8, 7.3Hz, 2H), 1.12 (t, J=7.2 Hz, 3H). ESI-MS: 342.2 [M+H]⁺. HPLC retentiontime: 12.812 min. HPLC purity 100%.

N-(1-(6-ethyl-4-oxo-1,4-dihydropyrimidin-2-yl)-3-methyl-1H-pyrazol-5-yl)-3-fluorobenzamide(12, AC10049) Isolated as a white solid (0.021 g, 0.062 mmol, 27%). ¹HNMR (500 MHz, DMSO-d₆) δ 12.17 (s, 1H), 8.02-8.00 (m, 1H), 7.92-7.90 (m,1H), 7.76-7.71 (m, 1H), 7.58-7.49 (m, 1H), 7.47 (s, 1H), 6.79 (s, 1H),2.93 (q, J=7.6 Hz, 2H), 2.27 (s, 3H), 1.27 (t, J=7.6 Hz, 3H). APCI-MS:342.3 [M+H]⁺. HPLC retention time: 13.607 min. HPLC purity 95.4%.

N-(1-(6-ethyl-4-oxo-1,4-dihydropyrimidin-2-yl)-3-methyl-1H-pyrazol-5-yl)cyclopropanecarboxamide(13, AC10050). Isolated as a white solid (0.018 g, 0.063 mmol, 34%). ¹HNMR (500 MHz, DMSO-d₆) δ 11.90 (s, 1H), 6.58 (s, 1H), 6.27 (s, 1H), 2.67(q, J=7.6 Hz, 2H), 2.21 (s, 3H), 1.83-1.78 (m, 1H), 1.23 (t, J=7.4 Hz,3H), 0.95-0.89 (m, 4H). APCI-MS: 286.0 [M−H]⁻. HPLC retention time:12.789 min. HPLC purity 98.5%.

N-(3-methyl-1-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)-1H-pyrazol-5-yl)cyclopropanecarboxamide(14, AC10051). Isolated as a white solid (0.025 g, 0.091 mmol, 38%). ¹HNMR (500 MHz, DMSO-d₆) δ 11.91 (s, 1H), 6.59 (s, 1H), 6.30 (s, 1H), 2.37(s, 3H), 2.22 (s, 3H), 1.86 (bs, 1H), 0.95-0.90 (m, 4H). APCI-MS: 274.1[M+H]⁺. HPLC retention time: 10.467 min. HPLC purity 99.1%.

3-fluoro-N-(3-methyl-1-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)-1H-pyrazol-5-yl)benzamide(15, AC10057). Isolated as a white solid (0.010 g, 0.031 mmol, 21%). 1HNMR (500 MHz, DMSO-d₆) δ 12.27 (s, 1H), 8.02-8.00 (m, 1H), 7.92-7.89 (m,1H), 7.82-7.80 (m, 1H), 7.75-7.70 (m, 1H), 7.57-7.48 (m, 1H), 7.45 (s,1H), 6.79 (s, 1H), 2.67 (s, 3H), 2.27 (s, 3H). APCI-MS: 326.1 [M−H]⁻.HPLC retention time: 12.739 min. HPLC purity 97.2%.

4-chloro-N-(1-(6-ethyl-4-oxo-1,4-dihydropyrimidin-2-yl)-3-methyl-1H-pyrazol-5-yl)benzamide(16, AC10058) Isolated as a white solid (0.010 g, 0.028 mmol, 15%). 1HNMR (500 MHz, DMSO-d₆) δ 12.68 (s, 1H), 8.02 (t, J=7.0 Hz, 2H), 7.45 (t,J=8.6 Hz, 2H), 6.76 (s, 1H), 6.30 (s, 1H), 2.66 (q, J=7.5 Hz, 2H), 2.27(s, 3H), 1.18 (t, J=7.5 Hz, 3H). APCI-MS: 358.2 [M+H^(]+). HPLCretention time: 12.718 min. HPLC purity 97.7%.

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Theimplementations should not be limited to the particular limitationsdescribed. Other implementations may be possible.

While the inventions have been illustrated and described in detail inthe drawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly certain embodiments have been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected.

It is intended that that the scope of the present methods andcompositions be defined by the following claims. However, it must beunderstood that this disclosure may be practiced otherwise than isspecifically explained and illustrated without departing from its spiritor scope. It should be understood by those skilled in the art thatvarious alternatives to the embodiments described herein may be employedin practicing the claims without departing from the spirit and scope asdefined in the following claims.

What is claimed is:
 1. A method for treatment of pain, opioiddependence, alcohol use disorder, or autism comprising the step ofadministering to a mammal in need of relief from pain or opioiddependence thereof a therapeutically effective amount of one or morecompounds of formula (I)

or a pharmaceutically acceptable salt thereof, and one or more carriers,diluents, or excipients, wherein R₁ is hydrogen, halo, hydroxy, amino,nitro, cyano, thio, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl, cycloalkyl, heterocyloalkyl, cycloalkenyl,heterocycloakenyl, heterocyclyl, alkoxyl, alkylamino, alkylthio,haloalkyl, sulphonyl, sulphonamide, alkylsulphonamide, or an optionallysubstituted aryl, arylalkyl, or arylalkenyl; R₂ is hydrogen, halo,hydroxy, amino, nitro, cyano, thio, alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyloalkyl,cycloalkenyl, heterocycloakenyl, heterocyclyl, alkoxyl, alkylamino,alkylthio, haloalkyl, sulphonyl, sulphonamide, alkylsulphonamide, or anoptionally substituted aryl, arylalkyl, or arylalkenyl; or R₁ and R₂ aretaken together with the attached carbons to form an optionallysubstituted cycle or heterocycle; R₃ is hydrogen, alkyl, alkenyl,alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl,heterocyloalkyl, cycloalkenyl, heterocycloakenyl, heterocyclyl, or anoptionally substituted aryl, arylalkyl, or arylalkenyl; R₄ is hydrogen,alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,cycloalkyl, heterocyloalkyl, cycloalkenyl, heterocycloakenyl,heterocyclyl, or an optionally substituted aryl, arylalkyl, orarylalkenyl; or R₃ and R₄ are taken together with the attached carbonsto form an optionally substituted cycle or heterocycle; and R₅ is analkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,cycloalkyl, heterocyloalkyl, cycloalkenyl, heterocycloakenyl,heterocyclyl, or an optionally substituted aryl, arylalkyl, orarylalkenyl.
 2. The method according to claim 1, wherein R₁ is hydrogen,a C₁-C₁₂ alkyl, or an optionally substituted C₃-C₁₂ cycloalkyl.
 3. Themethod according to claim 1, wherein R₂ is hydrogen, a C₁-C₁₂ alkyl, oran optionally substituted C₃-C₁₂ cycloalkyl.
 4. The method according toclaim 1, wherein R₁ and R₂ together with the attached carbons are linkedto form an optionally substituted cycle.
 5. The method according toclaim 1, wherein R3 is a C₁-C₁₂ alkyl.
 6. The method according to claim1, wherein R₄ is hydrogen.
 7. The compound according to claim 1, whereinR₅ is an optionally substituted C₄-C₁₂ heterocycle, aryl, arylalkyl, orarylalkenyl.
 8. The method according to claim 1, wherein the compound isselected from the group consisting of


9. The method of claim 1, wherein said pain is chronic pain.
 10. Themethod of claim 1 wherein treating pain or opioid dependence furthercomprises administering the compound of formula I in combination with anopioid drug, wherein the compound of Formula I enhances μ-opioidreceptor inhibition of adenylyl cyclase
 1. 11. The method of claim 10,wherein the opioid drug is selected from the group consisting ofcodeine, morphine, thebaine, oripavine, diacetylmorphine, nicomorphine,dipropanoylmorphine, diacetyldihydromorphine, acetylpropionylmorphine,desomorphine, methyldesorphine, dibenzoylmorphine, dihydrocodeine,ethylmorphine, heterocodeine, buprenorphine, etorphine, hydrocodone,hydromorphone, oxycodone, oxymorphone, fentanyl, alphamethylfentanyl,alfentanil, sufentanil, remifentanil, carfentanyl, ohmefentanyl,pethidine (meperidine), ketobemidone, desmethylprodine (MPPP),allylprodine, prodine, phenethylphenylacetoxypiperidine (PEPAP),promedol, propoxyphene, dextropropoxyphene, dextromoramide, bezitramide,piritramide, methadone, dipipanone, levomethadyl acetate (LAAM),difenoxin, diphenoxylate, loperamide, dezocine, pentazocine,phenazocine, buprenorphine, dihydroetorphine, etorphine, butorphanol,nalbuphine, levorphanol, levomethorphan, lefetamine, menthol,meptazinol, mitragynine, tilidine, tramadol, tapentadol, eluxadoline,nalmefene, naloxone, and naltrexone.
 12. A compound having a formula

or a pharmaceutically acceptable salt thereof, wherein R₁ is hydrogen,halo, hydroxy, amino, nitro, cyano, thio, alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyloalkyl,cycloalkenyl, heterocycloakenyl, heterocyclyl, alkoxyl, alkylamino,alkylthio, haloalkyl, sulphonyl, sulphonamide, alkylsulphonamide, or anoptionally substituted aryl, arylalkyl, or arylalkenyl; R₂ is hydrogen,halo, hydroxy, amino, nitro, cyano, thio, alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyloalkyl,cycloalkenyl, heterocycloakenyl, heterocyclyl, alkoxyl, alkylamino,alkylthio, haloalkyl, sulphonyl, sulphonamide, alkylsulphonamide, or anoptionally substituted aryl, arylalkyl, or arylalkenyl; or R₁ and R₂ aretaken together with the attached carbons to form an optionallysubstituted cycle or heterocycle; R₃ is hydrogen, alkyl, alkenyl,alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl,heterocyloalkyl, cycloalkenyl, heterocycloakenyl, heterocyclyl, or anoptionally substituted aryl, arylalkyl, or arylalkenyl; R₄ is hydrogen,alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,cycloalkyl, heterocyloalkyl, cycloalkenyl, heterocycloakenyl,heterocyclyl, or an optionally substituted aryl, arylalkyl, orarylalkenyl; or R₃ and R₄ are taken together with the attached carbonsto form an optionally substituted cycle or heterocycle; and R₅ is analkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,cycloalkyl, heterocyloalkyl, cycloalkenyl, heterocycloakenyl,heterocyclyl, or an optionally substituted aryl, arylalkyl, orarylalkenyl.
 13. The compound according to claim 12, wherein R₁ ishydrogen, a C₁-C₁₂ alkyl, or an optionally substituted C₃-C₁₂cycloalkyl.
 14. The compound according to claim 12, wherein R₂ ishydrogen, a C₁-C₁₂ alkyl, or an optionally substituted C₃-C₁₂cycloalkyl.
 15. The compound according to claim 12, wherein R₁ and R₂together with the attached carbons are linked to form an optionallysubstituted cycle.
 16. The compound according to claim 12, wherein R₃ isa C₁-C₁₂ alkyl.
 17. The compound according to claim 12, wherein R₅ is anoptionally substituted C₄-C₁₂ heterocycle, aryl, arylalkyl, orarylalkenyl.
 18. The compound according to claim 12, wherein thecompound is selected from the group consisting of


19. A pharmaceutical composition comprising one or more compounds ofclaim 12, or a pharmaceutically acceptable salt thereof, together withone or more diluents, excipients or carriers.
 20. A pharmaceuticalcomposition comprising one or more compounds of claim 12, or apharmaceutically acceptable salt thereof, in combination with one ormore other compounds by the same or different mode of action, togetherwith one or more diluents, excipients or carriers.